The ADCP assay was adapted from Ackerman et al. 201182 (link). Briefly, antigen was biotinylated using sulfo-NHS LC-LC biotin, coupled to yellow-green fluorescent Neutravidin 1 μm beads (Invitrogen, #F8776) for 2 h at 37 ˚C and washed two times in 0.1% bovine serum albumin (BSA) in phosphate buffer saline (PBS). 10 μL/well of coupled beads were added to 96-well plates with 10 μL/well of diluted sample for 2 h at 37˚C to form immune complexes. After incubation, the immune complexes were spun down and the supernatant was removed. THP-1 cells were added at a concentration of 2.5 × 104 cells/well and incubated for 18 h at 37 ˚C. After incubation, the plates were spun down, the supernatant was removed, and cells were fixed with 4% paraformaldehyde (PFA) for 10 min. Fluorescence was acquired with a Stratedigm 1300EXi cytometer. Phagocytic score was calculated using the following formula: (percentage of fluorescent+ cells) * (the geometric mean fluorescence intensity (gMFI) of the fluorescent+ cells)/10,000.
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